蛋白质化学 Protein Chemistry. Content Introduction of protein Amino acids Protein Structure ...

蛋白质化学 Protein Chemistry

Content

Introduction of protein

Amino acids

Protein Structure

Protein Properties

Protein Isolation and Purification

I Introduction of Protein

Proteins are the most abundant biological mac

romolecules, occurring in all cells and all parts

of cells.

Proteins occur in great variety, ranging in size f

rom relatively small peptides to huge polymers

with molecular weights in the millions.

Proteins are dehydration polymers of amino acids, with

each amino acid residue joined to its neighbor by a s

pecific type of covalent bond (Peptide bond ，肽键 ).

All proteins are constructed from the same ubiquitous s

et of 20 amino acids.

1. Proteins and Amino acids

(1) Elements

C 、 H 、 O 、 N 、 P 、 S

The nitrogen content of proteins is 15-17% ，with an average of 16% ，

ie.1g N = 6.25g Pr. Crude Pr.% = N% 6.25

2. Chemical composition of proteins

(2) Chemical composition

Simple protein — Contain only amino acid residues.

Conjugated protein – Contain non-amino acid part.

(1) Based on shape

Globular protein—able to dissolve and crystallize

Fibrous protein--generally water-insoluble

(2) Based on chemical composition

Simple protein –e.g.lysozyme

Conjugated protein –e.g.hemoglobin

Glycoproteins, lipoproteins, metalloproteins

3. Classification of proteins

(3) Based on solubility

Albumin soluble in water ∶ Globulin salted out with ammonium sulfate ∶ Glutelin insoluble in water, dissolve in in ∶

acidified or alkaline solution

Gliadin insoluble in water, dissolve in ethanol ∶ Protamine approximately 80% arginine and ∶

strongly alkaline

Histone less alkaline than protamine ∶ Scleroprotein insoluble proteins of animal organs∶

(4) Based on function

Active protein (Enzyme and antibody)

Passive protein (Collagen and keratin)

4. Biological function of proteins

Morphological function

Physiology function

Nutritional function

Animal

（ 1 ） Individual level

Hair and skin (keratins ）

Bone and teeth (collagen ）

Digestive system Digesting enzymes

Blood

Antibody

（ 2 ） Organ level

（ 3 ） Cell level

Shape of cell

Supporting body

Structural protein

Collagen

Functional protein

II Amino Acids

1. Hydrolysis of proteins

Proteins can be hydrolyzed by acid, alkali and proteases and broken down to peptides and mixture of amino acids.

The resulting characteristic proportion of different amino acids, namely, the amino acid composition was used to distinguish different proteins before the days of protein sequencing.

2. Amino acids structural features

All natural proteins were found to be built from a repertoire of 20 standard -amino acids.

The 20 -amino acids share common structural features.

Each has a carboxyl group and an amino group (but one has an i

mino group in proline) bonded to the same carbon atom, designa

ted as the a-carbon.

Each has a different side chain (or R group, R=“Remainder of the

molecule”).

The -carbons for 19 of them are asymmetric (or chiral), thus bei

ng able to have two enantiomers. Glycine has no chirality.

The two enantiomers of amino acid :

D- forms and L- forms

Align carbon atoms with L-glyceraldehyde, the amino group is on the left.

The horizontal bonds project out of the plane of the paper, the vertical behind.

3. Classification of amino acids

Nonpolar, aliphatic (hydrophobic) amino acids Aromatic amino acids Polar, uncharged amino acids Negatively and positively charged

according to the properties of their R groups

Gly, G Ala, A Val, V Leu, L Met, M Ile, I

Aliphatic amino acids

Phe, F; Tyr, Y; Trp, W

Aromatic amino acids

They are jointly responsible for the light absorption of

proteins at 280 nm

Ser, S Thr, T Cys, C Pro, P Asn, N Gln, Q

Polar, uncharged amino acids

Asp ,Glu

Negatively and positively charged

Lys, K; Arg, R; His, H

4. Acids and Bases properties of Amino Acids

When a crystalline amino acid, such as alanine, is dissolved in water, it exists in solution as the dipolar ion, or zwitterion, which can act either as an acid (proton donor) or as a base (proton acceptor):

Isoelectric point of Amino Acids

pI ( 等电点） is the pH of an aqueous solution of an ami

no acid at which the molecules on average have no net charge.

An acidic amino acid pI=(pK1+pKR)/2

A basic amino acid pI=(pKR+pK2)/2

5. Chemical Reactions of Amino Acids

Amino groups can be acetylated or formylated

Carboxyl groups can be esterified

(1) Peptide formation

(2) Carboxylic Acid Esterification

Esterification of the carboxylic acid is usually conducted under acidic conditions

(3) Amine Acylation

The pH of the solution must be raised to 10 or higher so that free amine nucleophiles are present in the reaction system.

(4) Ninhydrin reaction

III Protein StructureFour Levels of Architecture in Proteins

1. Primary structure

Primary structure is normally defined by the sequence of peptide-bonded amino acids and locations of disulfide bonds.

including all the covalent bonds between amino acids .

The relative spatial arrangement of the linked amino acids is unspecified.

2. Secondary structures Secondary structure refers to regular, recurring a

rrangements in space of adjacent amino acid resid

ues in a polypeptide chain. The Peptide Bond Is Rigid and Planar

(1) -Helix

Four models of -helix

(a) right-handed α-helix.

(b) The repeat unit is a single turn of the helix, 3.6 residues.

(c) α-helix as viewed from one end.

(d) A space-filling model of α-helix.

Factors Affected α- helix stability

A. steric repulsion is minimized and hydrogen bonding is maximized so the helix is stable.

B. Amino Acid Sequence Affects α Helix Stability

The twist of an α-helix ens

ures that critical interactio

ns occur between an amino

acid side chain.

(2) β-pleated sheet

β conformation is the more extended conformation of the polypeptide chains.

Connect the ends of t

wo adjacent segments

of an antiparallel β ple

ated sheet.

(3) β- turn

(4) Random coil

A representation of the 3D structure of the myoglobin protein. Alpha helices are shown in colour, and random coil in white, there are no beta sheets shown.

αhelix βsheet βturn

Random coil

Protein super-secondary structure

3. Tertiary structure

Tertiary structure refers to the spatial relation

ship among all amino acids in a polypeptide; it is the

complete three-dimensional structure of the polypep

tide.

Globular proteins can incorporate several types of secondary structure in the same molecule. Enzymes Transport proteins Peptide hormones Immunoglobulins

4. Quaternary Structure

The arrangement of proteins and protein subunits ( 亚单位 ) in three-dimensional complexes constitutes quaternary structure.

The interactions between subunits are stabilized and guided by the same forces that stabilize tertiary structure: multiple noncovalent interactions.

X-Ray Analysis Revealed the Complete Structure of Hemoglobin （血红蛋白）

5. Factors Affecting Protein Structure

1. Hydrogen bond ( 氢键 )

2. Electrostatic interaction ( 离子键 )

3. Hydrophobic interaction ( 疏水相互作用 )

4. van der waals force ( 范德华力 )

5. Disulfide bond ( 二硫键 )

A. 三级结构中的作用力

1. Disulfide bond 2. Electrostatic interaction

3. Hydrogen bond 4. Hydrophobic interaction

Primary structure determines secondary, tertiary and quaternary structures

Primary structure

S-S

6. Relationship between all grades structure

Conformational Changes in Hemoglobin Alter Its Oxygen-Binding Capacity

7. Relationship between structure and function of proteins

IV Protein Properties

Isoelectric point of protein

Colloidal properties

Protein denaturation

Protein precipitation

Protein sedimentation

Protein hydrolysis

Color reaction

UV light absorption

1. Isoelectric point of protein Acidic groups of Amino acids∶

γ-COOH group of Glu β-COOH group of Asp Phenolic hydroxy group of Tyr -SH group of Cys

Basic groups of Amino acids ∶ ε-NH2 group of Lys Imidazolyl group of His δ-guanidino group of Arg

Isoelectric point, pI, is the pH of an aqueous solution of an amino acid (or protein) at which the molecules on average have no net charge. 。

Proteins exist as zwitterions

PrNH3

+

COOHPr

NH3+

COO-Pr

NH2

COO-

OH-

H +

OH-

H +

¼æÐÔÀë×ÓpH=pI

ÑôÀë×ÓpH<pI

ÒõÀë×ÓpH>pI

The Isoionic point is the pH value at which a zwitterion molecule has an equal number of positive and negative charges.

pI is the pH value at which the net charge of the molecule, including bound ions is zero.

Whereas the isoionic point is at net charge zero in a deionized solution.

pI and isoionic point ( 等离子点 )

2. Colloidal properties

Solution （ < 1 nm ） Colloid （ 1 – 100 nm ） Suspension (> 100 nm)

Protein Molecular weight of 10,000-1000,000 Particle size of 2~20 nm Protein solution has colloidal properties.

Factors affecting the stability of protein colloidal solution

Polar surfaces pH ≠pI Same net charges on protein surface

Repulsion among protein molecules

Hydration water layer Charged amino acid residues

Water binding capacity of protein

Polar surfaces and water hydration layer of proteins

+

+

+

+

+

++

带正电荷的蛋白质

－ －－

－－

－－

－

带负电荷的蛋白质在等电点的蛋白质

Acid Alkaline

（ 1 ） Protein denaturation

Subtle changes in structure are usually regarded as “conformational adaptability”

Major changes in the secondary, tertiary, and quaternary structures without cleavage of backbone peptide bonds are regarded as “denaturation”.

3. Protein denaturation

（ 2 ） Reversibility of protein denaturation

（可逆性） Reversible

The proteins can regain their native state when the denaturing influence is removed.

Irreversible

Renaturation

Native State

Denaturation Urea （尿素）、 β-mercaptoethanol （巯基乙醇）

Renaturation( 复性） Remove Urea 、 β-ME

Unfolded State

（ 3 ） Denaturing agents

Physical agents Heat

The temperature at the tran

sition midpoint, where the c

oncentration ratio of native

and denatured states is 1, i

s known either as the meltin

g temperature Tm.

Hydrostatic pressure

Shear

Chemical agents pH and denaturation

Proteins are more stable against denaturation at their

isoelectric point than at any other pH. At extreme pH values, strong intramolecular electrost

atic repulsion caused by high net charge results in swelling and unfolding of the protein molecule.

Organic solvents and denaturation

Detergents and denaturation

Chaotropic Salts and Denaturation

（ 4 ） Changes in physical and chemical properties during protein denaturation

For most proteins, as denaturant concentration is increased, the value of y remains unchanged initially, and above a critical point its value changes abruptly from yN to yD.

（ 5 ） Application of protein denaturation

In favor of denaturation Sterilization with alcohol High pressure pasteurization

Prevention of denaturation Storage at low temperature

Replacement

4. Allosteric effect Hemoglobulin

Once the first heme-polypeptide subunit binds an O2

molecule, the remaining subunits respond by greatly increasing their oxygen affinity. This involves a change in the conformation of hemoglobin.

5. Precipitation of proteins

Changes in environmental conditions of protein colloidal solution might damage the hydration layer and surface charges and result in precipitation of proteins.

Salting-in ( 盐溶）

盐溶

蛋白质分子在等电点时，容易互相吸引，聚合沉淀；加入盐离子会破坏这些静电相互作用，使分子散开而溶于水

盐析Salting out （盐析）

蛋白质分子表面的疏水区域，聚集了许多水分子，盐浓度高时，这些水分子被盐抽出（水化层被破坏），暴露出的疏水区域，它们发生相互作用而沉淀。

（ NH4 ） 2SO4

6.Protein sedimentation Sedimentation is the tendency for molecules in solution to settle out of the fluid. This is due to their motion in response to the forces acting on them: gravity, centrifugal acceleration or electromagnetism.

60000~80000 转 /分

重力 60 万～ 80 万倍

7.Protein hydrolysis Splits the peptide bonds to give smaller p

eptides and amino acids. Occurs in the digestion of proteins. Occurs in cells when amino acids are nee

ded to synthesize new proteins and repair tissues.

8. Color reaction of protein

Color reaction of amino acids

Special color reaction of proteins Biuret protein assay

A chemical test for proteins

Biuret reagent is usually blue but turns violet wh

en it comes in contact with protein or a substanc

e with peptide bonds.

9. UV absorption of protein

Trp, Tyr and Phe

are responsible fo

r the light absorpt

ion of proteins at

280 nm.